Buffer exchange free, highly multiplexed bio-imaging with in situ DNA strand displacement

Abstract

Multiplexed fluorescent imaging methods are essential for resolving cellular transcriptional state with high resolution spatial information, but multiplexity is mostly limited to low numbers because of spectral overlap between the fluorophores that can be used. Although sequential fluidic exchange of DNA imagers expands its multiplexity, it requires time-consuming workflows and complex instrumentation. To eliminate such problems, here the toehold-FISH method as well as pre-programmed DNA probes is introduced, which is a novel imaging method that uses rapid and orthogonal DNA strand displacement reactions to enable highly multiplexed RNA imaging without buffer exchange steps and necessary accessories. In toehold-FISH, signal switching from one RNA target to the next is achieved by strand displacement reactions with sequentially added non- fluorescent DNA displacer strands, which take less than 30 seconds to complete signal switching in fixed cells. Because of vast sequence design space of DNA probes, unlimited multiplexity can be achieved.

Description

[0001] Attorney Docket no. 10457-596PC0

[0002] Buffer exchange free, highly multiplexed bio-imaging with in situ DNA strand displacement

[0003] BACKGROUND

[0004] Biological tissues, composed of millions of cells, maintain their functions through specific spatial organization and intercellular molecular signal exchanges. Understanding cellular organization at the molecular basis with single-cell level across entire tissues is crucial for elucidating tissue functions and disease mechanisms1-3. Single-cell spatial transcriptional profiling provides detailed molecular insights of cell states with spatial location information in the tissue context, enabling the classification of cell types4and deciphering cell-cell communications within tissue samples5, 6.

[0005] Techniques such as fluorescence in situ hybridization (FISH) imaging, allowing direct visualization of RNA in its intact biological environment, has been widely used to reveal critical details about the abundance and spatial arrangement of cellular RNA transcripts7, 8. However, to comprehensively study physiological states and biological functions of cells within their tissue context, high-dimensional molecular information must be resolved using high-plex fluorescence imaging to target numerous biomolecules. Consequently, advanced high-plex imaging techniques are highly desired for multiplexed transcriptional profiling.

[0006] Multiplexed fluorescent RNA imaging requires unambiguous fluorescent signal switching from one RNA target to the next. Conventional multiplexed fluorescence imaging uses different fluorophore-labeled probes to target multiple biomolecules, but the multiplexity is limited by spectral overlap of fluorophore, restricting multiplexing to 3-5 targets. Although hyperspectral properties of different dyes can be used for higher-plex imaging, more sensitive detectors, precise signal calibrations, and complex analysis algorithm arc needed to resolve the signals from different dyes9-11.

[0007] This limitation was overcome by sequential imaging methods using iterative labeling and imaging12-15, giving unlimited multiplexity theoretically. More recently, DNA-bascd barcoding and fluidic-exchange have enabled high-plex RNA imaging by staining all targets with DNA- barcoded in situ hybridization (ISH) probes in one round, followed by iterative binding and washing of fluorescently labeled oligonucleotides (DNA imagers)16-24. By encoding signals combinatorially across multiple rounds of DNA imager exchange, the multiplexing of FISH was Attorney Docket no. 10457-596PC0 exponentially increased, enabling spatially resolved, transcriptomic-scale RNA imaging25’31. Fluidic exchange of DNA imagers has become the dominant signal switching mechanism for the current imaging based spatial tran scrip tomics, with wide applications in neuroscience6, oncology32, and immunology33’35.

[0008] Despite its success, fluidic-exchange DNA imager methods face two major challenges: (1) buffer exchange steps can take tens of minutes to hours to have sufficient signal binding for the current RNA target and thorough signal removal for last round RNA targets, significantly slowing signal switching between rounds, and (2) the required fluidic devices are complex, costly, and prone to issues like air bubbles and tubing failures. Although commercial platform is available, they are generally very costly for acquisition and maintenance. These time-consuming washing steps, instrumentation complexities, and economic cost limit the widespread adoption of high-plex RNA imaging.

[0009] To address these challenges, the recently developed DNA thermal-plex36technique eliminated fluidic-exchange steps for multiplexed imaging by using temperature-controlled melting to sequentially and rapidly activate DNA thermal probes in situ. However, this method is limited to five thermal channels, profiling only 15 targets when combined with 3 fluorophore channels. Additionally, it requires a specialized heating chamber and temperature control module, still adding instrumentation complexity. Thus, there remains a need for a simpler, faster, and more accessible platform for high-plex RNA imaging for cell and tissue transcriptional profiling.

[0010] SUMMARY

[0011] Here is introduced toehold-FISH, a simple and highly multiplexed RNA imaging method for multiplexed spatial transcriptional profiling in cells and tissues. The method uses rapid, orthogonal toehold-mediated DNA displacement reactions to switch fluorescent signals from one RNA target to the next without fluidic exchange. DNA strand displacement allows a single stranded DNA invader to displace out a single stranded DNA from a DNA duplex through a toehold, with reaction kinetics being controlled through the DNA sequences37.

[0012] In toehold-FISH, novel toehold probes are designed to indirectly label RNA targets with fluorescent dyes. The toehold probes are composed through the hybridization of a fluorophore- conjugated imager strand and a quencher-conjugated quencher strand, enabling them to bind target Attorney Docket no. 10457-596PC0

[0013] RNAs through in situ hybridization (ISH) probes in a single step. The signal activation and removal arc driven by non-fluorcsccnt quencher and imager displacers added to the sample on- microscope, respectively. Displaced imager / imager displacer and quencher / quencher displacer DNAs remain at low concentrations (picomolar), rendering their presence negligible in the imaging buffer and eliminating the need of washing with fluidic exchange.

[0014] Displacement reactions are designed through simplified reactions pathways and optimal sequences to achieve rapid reaction rate for signal switch from one target to the next. Multiple rounds of signal switching can be achieved through consecutive additions of displacer strands, streamlining the workflow and reducing instrumentation complexity. Each round of signal switching in toehold-FISH is completed in under 30 seconds in fixed cells, and no special external accessories are required for on-microscope imaging beyond a standard fluorescence microscope and a general lab set-up.

[0015] A set of 25 different toehold probes with rapid kinetics and high orthogonality was developed, enabling toehold-FISH to achieve 25-plex RNA imaging in fixed cells within 20 minutes using a single fluorophore channel. Also, toehold-FISH ’s robust performance was demonstrated in complex heterogenous tissues by imaging 24 different RNAs in mouse retinal tissue to resolve different cell types.

[0016] This method significantly simplifying high-plex RNA imaging process with preprogrammed DNA probes. Toehold-FISH offers a rapid and efficient fluorescent switching method from one target to the next target with unlimited multiplexity for spatial transcriptional profiling in cells and tissues.

[0017] In some embodiments, DNA toehold probes for fluorescence in situ hybridization (FISH) imaging (toehold-FISH probes or TPs) are provided. The toehold-FISH probe comprises an imager strand and a quencher strand, and the imager strand binds to a unique nucleotide sequence (barcode domain c) appended to the 3’ end (or 5’-end) of an in situ hybridization probe (ISH probe) specific for a target RNA or DNA.

[0018] The imager strand has three domains, from the 5’ to 3’ (or 3’ to 5’), b*, c*, and d*. The b* domain hybridizes to the quencher strand, and at its 5’ end (or 3’ end) is labeled with a fluorophore. Attorney Docket no. 10457-596PC0

[0019] The c* domain hybridizes to the DNA barcode of an ISH probe specific for a target RNA or DNA; and the d* domain is a single-stranded toehold region for an imager displacer strand.

[0020] The quencher strand has two domains, from the 5’ to 3’ (or 3’ to 5’), a and b. The b domain hybridizes to the b* domain of the imager strand, and at its 3’ end (or 5’ end) is conjugated with a quencher quenching the fluorophorc of the imager strand. The a domain is a single- stranded toehold region for a quencher displacer strand.

[0021] When using the TPs, the fluorescence of the imager strand is detected when the quencher strand is removed from the imager strand by strand displacement initiated at the toehold region (a domain) of the quencher strand by a quencher displacer strand, releasing the quencher strand / quencher displacer hybridization fragment into the buffer. The quencher displacer strand has two domains (b*, a*), which are complementary to the two domains (b, a) of the quencher strand

[0022] Then, the fluorescence of the imager strand is removed from the target when the imager strand is removed by strand displacement initiated at the toehold region (d* domain) of the imager strand by an imager displacer strand, releasing the imager strand / imager displacer into the buffer. The imager displacer strand has two domains (d, c), which are complementary to the two domains (d*, c*) of the imager strand.

[0023] In certain embodiments, all domains of the aforementioned TPs have a nucleotide length in a range of 16-24 nucleotides, optionally about 18 nucleotides.

[0024] In certain embodiments, the fluorophore conjugated to the 5’ end (or 3’ end) of the imager strand is one selected from Atto-647, Atto-565, Atto-488, or Alexa-647. In certain embodiments, the quencher conjugated to the 3’ end (or 5’ end) of the quencher strand, which quenches the selected fluorophore, is BHQ-3, BHQ-2, Iowa black RQ, or Iowa black FQ.

[0025] In some embodiments, a kit comprising a TP and its matching quencher displacer / imager displace pair is provided. That is, the kit comprises imager strands labeled with a fluorophore, quencher strands, imager displacer strands, quencher displacer strands, and a small brochure introducing a matching barcode DNA sequence. Attorney Docket no. 10457-596PC0

[0026] In certain embodiments, a package comprising multiple of the aforementioned kit is provided. In the package comprising at least two TPs, the fluorophorc of each imager strand can be the same or a different fluorophore, and the package may further comprise a concentrated imaging buffer solution to be 5-50 times diluted for use.

[0027] In another embodiment, multiplexed tochold-FISH imaging method is provided, which depends on orthogonal toehold-mediated DNA displacement reactions to switch on and off fluorescent signals of a target RNA or DNA without fluidic exchange. The method comprises steps of i. fixing and permeabilizing a sample, which can be cells cultured on a coverslip or thin- sliced paraffin-embedded tissues; ii. labeling multiplex RNA or DNA targets with multiplex ISH probes, wherein each ISH probe has non-hybridizing unique barcode sequence (16-24 nucleotides, optionally about 18 nucleotides) at one end; e.g., adding mixture of ISH probes (20-40 nucleotides) to the sample, denaturing RNA and / or DNA in the sample at about 55-65 °C for 1-5 min, incubating the sample at about 45-55°C overnight for ISH probe / target hybridization, and rinsing the sample with a washing solution to remove unbound ISH probes; iii. assembling toehold-FISH probes, i.e., mixing quencher strands and imager strands (one set / tube) at 20-30°C, optionally about 25°C for 10-40 minutes, optionally about 30 minutes; iv. labeling multiplex barcodes with multiplex toehold-FISH probes, i.e., adding the assembled toehold-FISH probes to the sample, incubating the sample at room temperature for about 30 min, and rinsing the sample with a washing solution to remove unbound toehold-FISH probes; v. performing DAPI nuclei staining; vi. washing the sample, adding imaging buffer to the sample, and setting up the sample on the microscope stage; vii. measuring the basal level of fluorescence of the sample; viii. adding a quencher displacer strand of a first toehold-FISH probe to the sample; ix. after incubating the sample for a few minutes (1-5 minutes) at 20-30°C, measuring fluorescence for the target; x. adding an imager displacer strand of the first toehold-FISH probe to the sample; Attorney Docket no. 10457-596PC0 xi. repeating the steps of vii to x and determining the level of fluorescence by subtracting the basal level of fluorescence measured at step vii from the fluorescence measured at step ix; or optionally in the middle round n between the first round and the last round, the quencher displacer of the nth target (step viii of the nth round), and the imager displacer of the (n-7)th target (step x of the (n-J)th round) can be added together as a mixture (parallel displacement reactions), and in such a case, the mixture for first round contains only the quencher displacer for the first toehold probe, and the mixture for the final round contains only the imager displacer for the last toehold probe, and in step xi, the fluorescence signal for each target is determined by subtracting the basal level of fluorescence measured at the first round of step vii from the fluorescence measured at all round step ix.

[0028] BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1. The scheme of toehold-FISH. (a) The designed molecular reactions for toehold- FISH. The RNA target is labeled with DNA barcode domain c, and the toehold probe is formed by the hybridization of an imager and a quencher DNA strand through domains b* and b. The toehold probe binds to the target through the barcode, leaving single-stranded domains a and d* as toeholds for fluorescence signal activation and removal. To activate the signal, the quencher displacer initiates the strand displacement through domain a and displace the quencher from the target. Imaging can be acquired after signal activation. To eliminate the signal, the imager displacer will react with the imager to displace it from the target, (b) Scheme of the experimental setup. After the toehold probe binds to the barcode of ISH probes binding to a target, the quencher and imager displacer are sequentially directly added to the sample with pipetting to initiate signal activation and removal. No external accessories are needed; (c) The scheme of multiplexed imaging with different rounds of DNA strand displacements without washing steps exemplified with three targets. (1) Three different RNA targets (Tl, T2, and T3) are labeled with three different barcodes (cl, c2, and c3); (2) Three different toehold probes bind to their assigned barcodes of ISH probes binding to their RNA targets; (3 - 1 ) To activate the fluorescent signal for the first target, the quencher displacer 1 is added, followed by image acquisition; (3-2) The imager displacer for toehold probe 1 and quencher displacer for probe 2 arc added simultaneously to remove signal of Tl and activate signal of T2; (3-3) The imager displacer for toehold probe 2 and quencher displacer for probe 3 are added simultaneously to remove signal of T2 and activate signal of T3; (4) After Attorney Docket no. 10457-596PC0 all the rounds of imaging for each target, the overall images for all the targets are overlapped. All the displacer strands arc non-fluorcsccnt, and displacement imager and quencher arc in extremely low concentration. No fluidic exchange is needed to wash away the access of displacers or displaced imagers and quenchers.

[0030] Figure 2. The screening of sequences for rapid DNA displacement, (a) the workflow for the rapid sequence screening to design toehold probes, (b) The reaction design to screen the DNA sequence for displacement reactions using a universal imager and ISH probe for ELAVL1 mRNA. (c) The example of kinetic fitting for rapid and slow reactions, (d) The scattered plot for fitted rate constants of all 144 designed displacement reactions. The threshold is 1.2 x 105to select the sequences for rapid displacement reactions, (e) The example fluorescent images for rapid and slow reactions at different time points. All the scale bars are 10 pm.

[0031] Figure 3. The averaged RNA signal at different time points and kinetic fitting for all the designed 144 DNA displacement reactions for screening.

[0032] Figure 4. Validation of rapid signal activation and removal of toehold-FISH. (a) The scheme of RNA signal activation and removal of toehold-FISH. The added DNA displacer displaces out the quencher and imager DNA from the toehold probe bound with ISH probe on RNA targets to activate and remove signal, respectively; (b)(c)(d) The fluorescent imaging with toehold probe binding (b), after addition of quencher displacer (c), and after addition of imager displacer (d). (e)(f) the scheme of smFISH imaging and fluorescent images as positive control; (g)(i) The time course measurement of averaged RNA signal change after the addition of quencher displacer and imager displacer. Shaded regions denote mean ± s.d from all the RNA puncta in the field of view; (h)(j) Example images of a single RNA in situ at different time points after the addition of quencher displacer and imager displacer, respectively. The dimension of the images is 1 pm x 1 pm. (k) The violin plots of RNA puncta count after the toehold probe binding, addition of quencher displacer, addition of imager displacer, and smFISH positive control. The cell number used to count RNA copy number per cell for the samples were 58, 74, 61, and 73, respectively.

[0033] Figure 5. The secondary structure analysis of probe-barcode complex. NUPACK was used to analysis the minimal free energy state of the three-strand complex. Green dot=A, blue dot =C; black dot=G; and red dot=T Attorney Docket no. 10457-596PC0

[0034] Figure 6. The validation of 25 toehold probes in fixed cells, (a) The fluorescent imaging of RNAs with toehold probes, after addition of quencher displacer, and after the imager displacers. Scale bars, 10 pm. (b) The violin plots of resolved single cell RNA copy number after the toehold probe signal activation with the quencher displacer.

[0035] Figure 7. The validation of orthogonality for the designed 25 toehold probes. The fluorescent signal of probe and imager only was measured with the corresponding displacers, and non-cognate 48 other displacers. Non-cognate displacer mixtures didn’t induce any significant signal change, indicating high orthogonality between the displacement reactions.

[0036] Figure 8. Rapid 25-plex RNA imaging with toehold-FISH in fixed cells in a single fluorophore channel, (a) Schematic of the multiplexed imaging process using sequential strand displacement reactions. All 24 toehold probes are pre-assembled and labeled with Atto-565 fluorophores. The 24 distinct 1SH probes are hybridized with their RNA targets in situ, followed by the application of the 24 toehold probes for binding in one step. Quencher and imager displacer DNAs are sequentially added to the sample to complete the strand displacement for their corresponding RNA’s signal activation and removal. Fluorescent images were captured after each round of strand displacement, (b) Individual fluorescent images for the 24 RNA targets after each round of strand displacement in the white boxed region in (c). All the scale bars are 20 pm. (c) Composite images showing the 25-plex RNA overlap, (d) Single-cell RNA expression comparison between toehold-FISH (ntoehoid-nsH = 44 cells) and smFISH (nsmnsH > 30 cells for the statistics for all the 24 RNA targets) for 24 RNA targets. Error bars indicate the standard deviation of analyzed cells.

[0037] Figure 9. smFISH imaging for the selected RNA targets in U2OS cells, (a) The fluorescent imaging of selected 25 RNAs with smFISH. All the scale bars are 10 pm. (b) The violin plots of resolved single cell RNA expression level with smFISH for all the 25 RNA targets. N> 20 cells are used for statistical analysis.

[0038] Figure 10. The comparison of time consumption between the toehold-FISH and fluidic exchange method, (a) The heatmap of time consumption ratio of toehold-FISH to fluidic exchange; (b)(c)(d)(e)(f) The interface indicating the areas that toehold-FISH workflow is 2-fold, 5-fold, 10- fold, 20-fold, and 30-fold faster than fluidic-exchange workflow, respectively. Attorney Docket no. 10457-596PC0

[0039] Figure 11 . RNA imaging with toehold-FISH in mouse retina tissues, (a) The structures of retina tissue and selected Prcka mRNA markers located at the internuclear layer of the retinal tissue; (c)(d)(e) The fluorescent images for Prcka mRNA imaging after the toehold probe binding, quencher displacement, and imager displacement. The cell membrane was stained with WGA. All the scale bars are 10 pm. (f)(g) The time course measurement of single molecule RNA signal generation after the addition of quencher displacer and imager displacer. The reaction can be completed within 60s. Shaded regions denote mean ± s.d. with all the RNA puncta in the field of view, (f) The resolve single cell RNA copy number of Prcka mRNA with smFISH and toehold- FISH in retinal tissue samples. No significant difference is found between the two methods (nsm. FISH = 79 cells, n=toehold-FISH 45 cells).

[0040] Figure 12. Spatial profiling of mouse retinal tissue with 24-plex toehold-FISH RNA imaging in single fluorophore channel, (a) The organization of different cell types located in three different layers of retinal tissues; (b) The scheme of toehold-FISH for multiplexed imaging with 24 different toehold probes labeled with Atto-565 fluorophores; After all the toehold probe binding, non-fluorescent displacers are sequentially added to activate or removal RNA fluorescent signal via rapid orthogonal displacement reactions, (c) The fluorescent images of 24 RNAs in mouse retinal tissue after each round of toehold probe signal activation and removal for 1 min. The 24 RNAs were presented in 6 groups of 4 RNAs overlapping images. The cell membrane was stained with WGA. The boxed regions in each overlapped panel are also shown in d. All the scale bars are 20 pm. (d) The selected resolved different cell types in the retinal tissues and corresponding RNA markers. All the scale bars are 5 pm. (e) The violin plots of single cell expression level of 24 RNAs. N > 40 cells were used for statistic analysis for all the RNA markers.

[0041] Figure 13. Fluorescent images of individual 24 RNA target after each round of displacement reactions. All the 24 RNAs were shown in desired location in the retinal tissues. The scale bars are 20 pm.

[0042] Figure 14. Single cell expression of RNA with smFISH in in retinal tissues, (a) Fluorescent images of smFISH for the 24 RNA targets. The cell membrane was stained with WGA. All the scale bars are 20 pm. (b) Violin plots of the RNA expression level in the expressed cell types, n > 20 cells were used for the statistical analysis. Attorney Docket no. 10457-596PC0

[0043] DETAILED DESCRIPTION

[0044] 1. Overview

[0045] The toehold-FISH imaging method enables highly multiplexed RNA imaging in cells and tissues without fluidic exchange or additional accessories, utilizing strand displacement reactions.

[0046] By simply adding displacer strands to the sample imaging chamber using standard pipettes, rapid signal activation and removal are achieved through pre-programmed orthogonal DNA displacement reactions. This approach significantly streamlines the multiplexed imaging workflow by reducing signal switching time from tens of minutes or even hours, typical in previous fluidic exchange-based methods, to less than one minute. It supports on-scope imaging without requiring costly or complex instrumentation.

[0047] Increasing the number of signal exchange rounds is highly desirable in imaging-based spatial transcriptomics to achieve transcriptome coverage comparable to that of sequencing methods for untargeted, discovery-driven research53. However, as the number of signal exchange rounds increases, the fluidic exchange becomes more dominant in the overall workflow, and the multiplexed imaging process becomes significantly time-consuming and error prone.

[0048] To address this issue, a new method is introduced here. The workflow of toehold-FISH is increasingly faster compared with conventional fluidic exchange method when a higher number of exchange rounds is needed. To test this method are developed 25 different toehold probes for 25 rounds of displacement reactions in a single fluorophore for 25-plex imaging. The vast DNA sequence space theoretically allows for the development of hundreds of such probes to further meet the needs of high-multiplex imaging. Multiple spectrally separated fluorophores can be easily integrated with toehold-FISH to further increase the multiplcxity while maintaining workflow simplicity. Replacing the fluidic exchange of DNA imagers with DNA displacement reactions will significantly simplify future high-plex imaging-based spatial transcriptomics.

[0049] The toehold-FISH can be readily integrated with well-developed hydrogel embedding and tissue clearing54, 55to further facilitate reaction speed for tissue imaging by creating a more homogenized reaction environment. It was noticed that the tissue complexity may affect the signal switching of toehold-FISH, potentially because of slower diffusion of displacer strands within tissue due to their densely packed cells and extra- cellular matrix. Attorney Docket no. 10457-596PC0

[0050] Furthermore, these orthogonal and rapid toehold probes are not limited to multiplexed RNA imaging. They can be readily adapted for imaging other modalities by simply changing the binding entities that carry toehold probe barcodes, such as chromosomal DNA and proteins, in cell and tissue samples. Additionally, combinatorial encoding of toehold probes could scale RNA imaging to the transcriptome level.

[0051] Toehold-FISH for multiplexed imaging was demonstrated in thin tissue sections, and it can also be applied to thick tissues, potentially further reducing the weeks of long signal exchange times in multiplexed thick tissue imaging56, 57.

[0052] It can be envisioned that the toehold- FISH method will profoundly change signal switching in multiplexed fluorescent imaging, significantly enhancing the accessibility and usability of imaging-based spatial biology for broad applications.

[0053] The cost of toehold-FISH is also low because no additional accessories are required beyond a standard fluorescence microscope and DNA probes, with all expenses tied to DNA probes. The cost of toehold-FISH RNA imaging includes four components: a primary probe pool for RNA binding, toehold probe imagers, toehold probe quenchers, and displacer DNA strands. A standard pool of -48 unpurified probe oligonucleotides costs ~$200, yielding enough for 250 mL of 1 pM probe solution. An imager typically costs $200 for -10 nmole, producing 1 mL of 1 pM solution. A quencher costs $80 for -15 nmole, providing 1 .5 mL of 1 pM solution. A displacer strand costs $5 for 10 nmole, resulting in 200 pL of 100 pM solution. In a typical toehold-FISH RNA imaging experiment, -120 pL of ISH probe at 100 nM is used, costing -$0,012, while 120 pL of 200 nM DNA thermal probe costs ~$8 per experiment. Costs can be lowered by ordering unlabeled DNA oligonucleotides and performing fluorophore and quencher conjugation in-house.

[0054] 2. Definitions

[0055] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference. Attorney Docket no. 10457-596PC0

[0056] Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein, and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed through the present specification unless otherwise indicated.

[0057] The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of + / -10% or less, + / -5% or less, + / -! % or less, and + / -0.1 % or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.

[0058] Reference throughout this specification to “one embodiment”, “an embodiment,” “an example embodiment,” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” or “an example embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

[0059] As used herein, the term "target" refers to a specific DNA or RNA nucleotide sequence, which is to be identified with fluorescence signal. The target sequence is hybridized with a Watson-Crick base-pairing fragment, which is referred to as in situ hybridization probe (ISH probe) that is directly or indirectly labeled with a fluorophore molecule.

[0060] The term “nucleotide” as used herein refers to a subunit of a nucleic acid (whether DNA or RNA or an analogue thereof) which may include, but is not limited to, a phosphate group, a 5- carbon sugar group, and a nitrogen containing base, as well as analogs of such sub-units. Other Attorney Docket no. 10457-596PC0 groups (e.g., protecting groups) can be attached to the sugar group and nitrogen containing base group. It will be appreciated that, as used herein, the terms “nucleotide” will include those moictics which contain not only the naturally occurring purine and pyrimidine bases, e.g., adenine (A), thymine (T), cytosine (C), guanine (G), or uracil (U), but also modified purine and pyrimidine bases and other heterocyclic bases which have been modified (these moieties are sometimes referred to collectively, as “purine and pyrimidine bases and analogs thereof’).

[0061] As used herein, the term “nucleic acid” refers to any polyribonucleotide or polydeoxyribonucleotide that may be unmodified RNA or DNA or modified RNA or DNA. Thus, for instance, nucleic acid as used herein refers to, among others, single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double- stranded RNA, and RNA that is mixture of single- and double- stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or double-stranded, or a mixture of single- and doublestranded regions. The nucleic acid may comprise artificial nucleic acids including peptide nucleic acids (PNA), Morpholino and locked nucleic acids (LNA), as well as glycol nucleic acids (GNA), threose nucleic acids (TNA) and hexitol nucleic acids (HNA). Each of these is distinguished from naturally occurring DNA or RNA by changes to the backbone of the molecule.

[0062] The terms “nucleotide sequence,” or “nucleic acid sequence” as used herein refers to the specific order or arrangement of nucleotides (adenine, guanine, cytosine, and thymine in DNA, or uracil in RNA, represented using the letters A, C, G, and T (or U in RNA)) within a nucleic acid, DNA or RNA.

[0063] As used herein, the term “strand displacement” refers to an enzyme-free method for replacing one strand of nucleic acid such as RNA or DNA (output) with another strand (input). It is a process of exchanging two single strands utilizing the hybridization via Watson-Crick base pairing (A-T / U and C-G) (Yurke, Bernard (2000). "A DNA-fueled molecular machine made of DNA". Nature. 406 (6796): 605-8). Briefly, the first strand of double- stranded hybridization has an overhanging “toehold” region, which is complementary to a portion of a third strand, which is a single-stranded DNA (ssDNA). The process of strand displacement initiates at the toehold region by the hybridization of the third strand to the first strand, creating a DNA, RNA, or DNA / RNA complex composed of three strands, in which the second strand, which is the strand initially hybridized with the first strand, and the third strand each partially hybridize with the first strand. Attorney Docket no. 10457-596PC0

[0064] After the binding of the third strand to the toehold region of the first strand, branch migration occurs, allowing the displacement of the second strand by the third strand. The initial toehold binding of the third strand is an endothermic rate limiting step and can be helped by enhancing the hybridization strength at the toehold region, i.e., length and sequence of the participating strands.

[0065] As used herein, the term “in situ hybridization (ISH)” refers to a technique that is used for the localization of a specific nucleotide sequence of RNA or DNA in cells, tissue sections, and even whole tissue. This method is based on the complementary binding of a nucleic acid fragment (probe), to a specific target sequence of DNA or RNA, and usually a reporter molecule is attached to the probe. Visualization of the reporter molecule makes it possible to localize DNA or RNA sequences in cell or tissue samples. Unlimited examples of in situ hybridization probes comprises double-stranded DNA (dsDNA) probes, single-stranded DNA (ssDNA) probes, RNA probes (riboprobes), synthetic oligonucleotide probes (PNA, LNA). Unlimited examples of probe labeling materials comprise radioactive isotopes such as32P,35S, and3H and non-radioactive molecules such as biotin, digoxigenin, and fluorescent dye (FISH). ISH technique can be employed for the study in the field of microbiology (e.g., bacterial 16S rRNA), pathology (pathogen profiling, abnormal gene expression), developmental biology (gene expression profiling in embryonic tissues), karyotyping and phylogenetic analysis (unique FISH patterns on individual chromosomes, chromosomal aberrations), and physical mapping (mapping clones on chromosomes and direct assignment of mapped clones to chromosomal regions associated with heterochromatin or euchromatin). (ncbi.nlm.nih.gov / probe / docs / techish / )

[0066] As used herein, the term “DAPI staining” refers to DNA staining with a blue-fluorescent dye, 4',6-diamidino-2-phenylindole, which is excited at the violet wavelength range (405 nm). DAPI exhibits ~20-fold enhancement of fluorescence upon binding to AT regions of dsDNA, and is commonly used as a nuclear counterstain in fluorescence microscopy, flow cytometry, and chromosome staining for counting cells, measuring apoptosis, sorting cells based on DNA content, and nuclear segmentation in high-content imaging analysis. DAPI is generally used to stain fixed cells (dead cells) since the dye is cell impcrmcant. For nuclear counterstaining in live-cells, Hoechst 33342 dye is commonly used.

[0067] 3. Examples of Embodiment Attorney Docket no. 10457-596PC0

[0068] Herein is described a bio-imaging method that can detect multiplex RNA (or DNA) targets using a single wavelength fluorophorc without buffer change between each fluorescence detection step. First, multiplex RNA (or DNA) targets are hybridized with multiplex target specific ISH probes appended with individual DNA ‘barcodes’, respectively. Each barcode binds to a portion of an imager strand of a toehold probe (TP) that is composed of a fluorophore-imager strand and a quencher strand. After TP binding to the barcode of an ISH probe hybridizing to a specific target RNA (or DNA), for the detection of fluorescence, quencher displacer is added, which hybridizes to an end of the quencher strand, i.e., first toehold region, and a strand displacement reaction begins to bare the fluorophorc of the imager strand from the quencher of the quencher strand. Then, to remove the fluorescence signal from the target, imager displacer is added, which hybridizes to an end of the imager strand, i.e., second toehold region, and a strand displacement reaction begins to detach the fluorophore-imager strand from the barcode. The fluorophore-imager / imager displacer fragment is diffused away, and fluorescence signal of the target is off. Using this method, multiplex targets labeled with multiplex toehold probes respectively at the beginning can be detected one by one by sequential addition of a pair of quencher displacer and imager displacer without buffer change steps between each target fluorescence detection.

[0069] This method allows for “unlimited” targets to be detected. By designing probes comprising toeholds, it overcomes buffer exchange inconvenience and slide relocation problem without using an expensive perfusion / drainage system.

[0070] Compared with existing methods, the toehold FISH disclosed here will provide greater accessibility and be compatible with conventional microscope setups without sensitive detector calibration nor complex instrumentation. In addition, toehold FISH also has a much faster probe signal transition time compared with the buffer-exchange based devices because of rapid DNA strand displacement in situ, reducing the process time from hours to minutes.

[0071] This method depends on the design of a target- specific ISH probe with a unique DNA barcode, a barcode- specific DNA probe comprising a quencher strand and an imager strand, each of which comprises a toehold region, and two types of toehold binding DNA fragments, i.e., quencher displacer and imager displacer.

[0072] Through a meticulous sequence selection process described in EXAMPLES, nucleotide sequences with good strand displacement kinetics were selected for toehold-FISH probe (TP) Attorney Docket no. 10457-596PC0 design. TP1 to TP25 are unlimited examples of such designed probes. Any nucleotide sequences of any length, optionally 16-24 nucleotides, c.g., 18 nucleotides, with proper strand displacement kinetics can be considered for TP design.

[0073] In one embodiment, DNA toehold probe (TP1) is provided, which comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:51 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:26, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 101 and SEQ ID NO:76, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 1.

[0074] In another embodiment, DNA toehold probe (TP2) is provided, which comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:52 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:27, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 102 and SEQ ID NO:77, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 2.

[0075] In another embodiment, DNA toehold probe (TP3) is provided, which comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:53 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:28, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 103 and SEQ ID NO:78, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 3.

[0076] In another embodiment, DNA toehold probe (TP4) is provided, which comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:54 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:29, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 104 and SEQ ID NO:79, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 4.

[0077] In another embodiment, DNA toehold probe (TP5) is provided, which comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:55 and a quencher strand comprising a Attorney Docket no. 10457-596PC0 nucleotide sequence of SEQ TD NO:30, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 105 and SEQ ID NO:80, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 5.

[0078] In another embodiment, DNA toehold probe (TP6) is provided, which comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:56 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:31, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 106 and SEQ ID NO:81, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 6.

[0079] In another embodiment, DNA toehold probe (TP7) is provided, which comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:57 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:32, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 107 and SEQ ID NO:82, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 7.

[0080] In another embodiment, DNA toehold probe (TP8) is provided, which comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:58 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:33, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 108 and SEQ ID NO:83, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 8.

[0081] In another embodiment, DNA toehold probe (TP9) is provided, which comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:59 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:34, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 109 and SEQ ID NO:84, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 9. Attorney Docket no. 10457-596PC0

[0082] In another embodiment, DNA toehold probe (TP10) is provided, which comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:60 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:35, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 110 and SEQ ID NO:85, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 10.

[0083] In another embodiment, DNA toehold probe (TP11) is provided, which comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:61 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:36, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 111 and SEQ ID NO: 86, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 11.

[0084] In another embodiment, DNA toehold probe (TP12) is provided, which comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:62 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:37, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 112 and SEQ ID NO:87, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 12.

[0085] In another embodiment, DNA toehold probe (TP 13) is provided, which comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:63 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:38, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 113 and SEQ ID NO:88, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 13.

[0086] In another embodiment, DNA toehold probe (TP 14) is provided, which comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:64 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:39, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 114 and SEQ ID NO: 89, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 14. Attorney Docket no. 10457-596PC0

[0087] In another embodiment, DNA toehold probe (TP15) is provided, which comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:65 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:40, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 115 and SEQ ID NO:90, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 15.

[0088] In another embodiment, DNA toehold probe (TP 16) is provided, which comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:66 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:41, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 116 and SEQ ID NO:91, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 16.

[0089] In another embodiment, DNA toehold probe (TP17) is provided, which comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:67 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:42, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 117 and SEQ ID NO:92, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 17.

[0090] In another embodiment, DNA toehold probe (TP 18) is provided, which comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:68 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:43, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 118 and SEQ ID NO:93, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 18.

[0091] In another embodiment, DNA toehold probe (TP 19) is provided, which comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:69 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:44, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 119 and SEQ ID NO:94, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 19. Attorney Docket no. 10457-596PC0

[0092] In another embodiment, DNA toehold probe (TP20) is provided, which comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:70 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:45, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 120 and SEQ ID NO:95, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 20.

[0093] In another embodiment, DNA toehold probe (TP21) is provided, which comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:71 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:46, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 121 and SEQ ID NO:96, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 21.

[0094] In another embodiment, DNA toehold probe (TP22) is provided, which comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:72 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:47, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 122 and SEQ ID NO:97, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 22.

[0095] In another embodiment, DNA toehold probe (TP23) is provided, which comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:73 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:48, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 123 and SEQ ID NO:98, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 23.

[0096] In another embodiment, DNA toehold probe (TP24) is provided, which comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:74 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:49, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 124 and SEQ ID NO:99, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 24. Attorney Docket no. 10457-596PC0

[0097] In another embodiment, DNA toehold probe (TP25) is provided, which comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:75 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:50, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 125 and SEQ ID NO: 100, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 25.

[0098] For multiplex fluorescence measurement, multiplex probes are added to the sample at the beginning. After completing the first round of fluorescence measurement for the first target, the second round of fluorescence measurement for the second target is started by simply adding a second quencher displacer to turn on fluorescence of the second target, and then finished by adding a second imager displacer to turn off fluorescence. The third round of fluorescence measurement is started and finished in the same manner using a third pair of quencher displacer and imager displacer, and this sequential addition of a pair of displacer strands continues until the fluorescence measurement of the last target without any washing step between each target fluorescence measurement. The intensity of fluorescence signal for each target can be determined by subtracting the background fluorescence signal measured at the beginning of the experiment or measured after addition of imager displacer of the previous round.

[0099] While multiple RNA targets are tested below as examples for the application of toehold probes (TPs), multiple DNA target sequences can also be contemplated for the application of this new fluorescence microscopy method in the field of microbiology, pathology, developmental biology, genetics as well as taxonomy.

[0100] Also, multiple protein targets can be detected using the TPs if DNA barcode-conjugated antibodies against target proteins are available. Further, if DNA or RNA aptamers against target proteins, peptides, carbohydrates, small molecules, toxins, are available, the TPs can be applied to the sample in a very similar manner to the method described here.

[0101] It is convenient to detect the same emission wavelength of fluorescence for all the TPs applied to multiplex targets. However, if need be, multiplex fluorophores can be conjugated to multiplex TPs (imager strands) respectively. Unlimited examples of fluorophores that can be conjugated to the imager strand include; (probe name (excitation wavelength (nm) I emission wavelength (nm)); hydroxycoumarin (325 / 386), aminocoumarin (350 / 445), methoxycoumarin Attorney Docket no. 10457-596PC0

[0102] (360 / 410:), cascade blue ((375);401 / 423), pacific blue (403 / 455), pacific orange (403 / 551), lucifer yellow (425 / 528), NBD (466 / 539), R-Phycocrythrin (PE) (480;565 / 578), PE-Cy5 conjugates (480;565;650 / 670), PE-Cy7 conjugates (480;565;743 / 767), Red 613 (480;565 / 613), PerCP (490 / 675), TruRed (490,675 / 695), FluorX (494 / 520), Fluorescein (495 / 519), BODIPY-FL (503 / 512), TRITC (547 / 572), X-Rhodamine (570 / 576), Eissamine Rhodamine B (570 / 590), Texas Red (589 / 615), allophycocyanin (APC) (650 / 660), APC-Cy7 conjugates (650;755 / 767); Alexa Fluor dyes such as Alexa Fluor 350 (343 / 442), Alexa Fluor 405 (401 / 421), Alexa Fluor 430 (434 / 540), Alexa Fluor 488 (499 / 519), Alexa Fluor 500 (503 / 525), Alexa Fluor 514

[0103] (517 / 542), Alexa Fluor 532 (530 / 555), Alexa Fluor 546 (561 / 572), Alexa Fluor 555 (553 / 568), Alexa Fluor 568 (579 / 603), Alexa Fluor 594 (591 / 618), Alexa Fluor 610 (610 / 629), Alexa Fluor 633 (632 / 648), Alexa Fluor 647 (652 / 668), Alexa Fluor 660 (663 / 691), Alexa Fluor 680 (680 / 702), Alexa Fluor 700 (696 / 719), Alexa Fluor 750 (752 / 776), and Alexa Fluor 790 (782 / 804); Cy Dyes such as Cy2 (489 / 506), Cy3 ((512);550 / 570;(615)), Cy3B (558 / 572;(620)), Cy3.5 (581 / 594;(640)), Cy5 ((625);650 / 670), Cy5.5 (675 / 694), and Cy7 (743 / 767); DyEight dyes such as DyEight 350 (353 / 432), DyEight 405 (400 / 420), DyLight 488 (493 / 518), DyLight 549 (562 / 576), DyLight 594 (593 / 618), DyLight 633 (638 / 658), DyLight 649 (654 / 673), DyLight 680 (692 / 712), DyLight 750 (752 / 778), and DyLight 800 (777 / 794); Nucleic acid probes such as Hoechst 33342 (343 / 483), DAPI (345 / 455), Hoechst 33258 (345 / 478), S YTOX Blue (431 / 480), Chromomycin A3

[0104] (445 / 575), mithramycin (445 / 575), YOYO-1 (491 / 509), ethidium bromide (493 / 620), acridine orange (503 / 525), acridine yellow (470 / 550), (SYTOX Green (504 / 523), TOTO-1 or TO-PRO-1 (509 / 533), thiazole orange (510 / 530), propidium iodide (PI) (536 / 617), LDS 751 (543;590 / 712;607), 7-AAD (546 / 647), SYTOX Orange (547 / 570), TOTO-3 or TO-PRO-3 (642 / 661), and DRAQ5 (647 / 681,697); cell function probes such as Indo-1 ((361 / 330) / (490 / 405)), Fluo-3 (506 / 526), DCFH (505 / 535), DHR (505 / 534), and SNARF ((548 / 579) / (587 / 635); fluorescent proteins such as Y66H (360 / 442), Y66F (360 / 508), EBFP (380 / 440), EBFP2 (383 / 448), Azurite (383 / 447), GFPuv (385 / 508), T-Sapphire (399 / 511), TagBFP (402 / 457), cerulean (433 / 475), mCFP (433 / 475), ECFP (434 / 477), CyPet (435 / 477), Y66W (436 / 485), dKeima-Red (440 / 616), mKeima-Red (440 / 620), TagCFP (458 / 480), AmCyanl (458 / 489), mTFPl (Teal) (462 / 492), S65 A (471 / 504), Midoriishi-Cyan (472 / 495), wild Type GFP (396,475 / 508), S65C (479 / 507), TurboGFP (482 / 502), TagGFP (482 / 505), TagGFP2 (483 / 506), AcGFPl (484 / 510), S65L (484 / 510), Emerald (487 / 509), S65T (488 / 511), EGFP (488 / 507), Attorney Docket no. 10457-596PC0

[0105] Azami-Green (492 / 505), ZsGreenl (493 / 505), Dronpa-Green (503 / 518), TagYFP (508 / 524), EYFP (514 / 527), Topaz (514 / 527), Venus (515 / 528), mCitrinc (516 / 529), YPct (517 / 530), Turbo YFP (525 / 538), PhiYFP (525 / 537), PhiYFP-m (525 / 537), Zs Yellowl (529 / 539), mBanana (540 / 553), Kusabira-Orange (548 / 559), mOrange (548 / 562), mOrange2 (549 / 565), mKO (548 / 559), TurboRFP (553 / 574), tdTomato (554 / 581), DsRed-Express2 (554 / 591), TagRFP (555 / 584), DsRed monomer (557 / 592), DsRed2 ("RFP") (563 / 582), mStrawberry (574 / 596), TurboFP602 (574 / 602), AsRed2 (576 / 592), mRFPl (584 / 607), J-Red (584 / 610), mCherry (587 / 610), HcRedl (588 / 618), mKate2 (588 / 633), Katushka (TurboFP635) (588 / 635), mKate (TagFP635) (588 / 635), TurboFP635 (588 / 635), mPlum (590 / 649), mRaspberry (598 / 625), mNeptune (600 / 650), and E2-Crimson (611 / 646); and other probes including monochlorobimane (380 / 461), Calcein (496 / 517), and HyPer ((420 / 500) / 516). In addition, there are ATTO fluorescent dyes such as ATTO 390 (390 / 476), ATTO 425 (439 / 485), ATTO 430LS (436 / 545), ATTO 465 (453 / 506), ATTO 488 (500 / 520: Alexa 488, FITC, FAM), ATTO 490LS (495 / 658), ATTO 495 (498 / 526), ATTO 514 (511 / 532: Alexa 514), ATTO 520 (517 / 538: JOE, TET), ATTO 532 (532 / 552: Alexa 532, HEX(533 / 559)), ATTO Rho6G (533 / 557; HEX (533 / 559)), ATTO 542 (542 / 562), ATTO 550 (554 / 576: TAMRA (552 / 578), Cy3), ATTO 565 (564 / 590: Cy3.5, ROX (578 / 604)), ATTO Rho3B (566 / 589), ATTO Rholl (572 / 595: ROX (578 / 604)), ATTO Rhol2 (576 / 601), ATTO Thiol2 (582 / 607), ATTO RholOl (587 / 609), ATTO 590 (593 / 622: Alexa 594, Texas Red), ATTO 594 (603 / 626: Alexa 594), ATTO Rhol3 (603 / 627: Alexa 594), ATTO 610 (616 / 633), ATTO 620 (620 / 642), ATTO Rhol4 (626 / 646: Alexa 633), ATTO 633 (630 / 651: Alexa 633), ATTO 647 (647 / 667: Cy5, Alexa 647), ATTO 647N (646 / 664: Cy5, Alexa 647), ATTO 655 (663 / 680: Cy5, Alexa 647), ATTO Oxal2 (662 / 681), ATTO 665 (662 / 680), ATTO 680 (681 / 698: Cy5.5), ATTO 700 (700 / 716: Cy5.5), ATTO 725 (728 / 751), ATTO 740 (743 / 763). Also, boron dipyrromethene (BODIPY) fluorophore series can be conjugated to the imager strand, such as BODIPY 493 / 503 (493 / 503), BODIPY FL-X (504 / 510), BODIPY R6G-X (529 / 547), BODIPY 564 / 570 (563 / 569), BODIPY TMR-X (544 / 570), BODIPY 581 / 591 (581 / 591), BODIPY TR-X (588 / 616), BODIPY 630 / 650-X (625 / 640), BODIPY 650 / 665-X (646 / 660). In addition, there are other fluorophores for imager strand conjugation, comprising Marina Blue® (362 / 459), EDANS (336 / 468), coumarin (432 / 472), Dansyl (335 / 518), FAM (495 / 519), Oregon Green (500 / 520), Rhodamine Green-X (503 / 528), NBD-X (466 / 535), Yakima Yellow (531 / 549). VIC (538 / 554), Attorney Docket no. 10457-596PC0

[0106] Rhodamine 6G (524 / 557), Rhodamine Red-X (560 / 580), Redmond Red (579 / 595), Cal Red (583 / 603), and LC® Red 640 (625 / 640).

[0107] As for the quencher molecules for quencher strands, unlimited examples are BHQ (Black Hole Quenchers)-0® (absorbance maximum of 495 nm, range: 430-520 nm), BHQ-1® (absorbance maximum of 534 nm, rangc:480-580 nm), BHQ-2® (absorbance maximum of 579 nm, range:550-650 nm), BHQ-3® (absorbance maximum of 680 nm, range: 620-730 nm), DDQ1 (2,3-dichloro-5,6-dicyano-l,4-benzoquinone) (absorbance maximum of 440 nm, range: 380-550 nm), DDQII (absorbance maximum of 650 nm, range: 570-680 nm), DABCYL (4,4- dimethylaminoazobenzene-4-carboxylic acid) (absorbance maximum of 479 nm, range: 390-510 nm), EDQ® (Eclipse Dark Quencher) (absorbance maximum of 522 nm. range: 460-570 nm), QXL® 520 (absorbance maximum of 508-530 nm, range: 480-550 nm), QXL® 570 (absorbance maximum of 538-577 nm, range: 530-600 nm), QXL® 610 (absorbance maximum of 594-628 nm, range: 545-665 nm), QXL® 670 (absorbance maximum of 668 nm, range: 610-700 nm). IR- QXL® (absorbance maximum of 772 nm, range: 640-850 nm). QSY-35® (absorbance maximum of 475 nm, range: 410-500 nm ), QSY-7® (absorbance maximum of 560 nm, range: 500-600 nm), QSY-9® (absorbance maximum of 562 nm, range: 500-600 nm), QSY-21® (absorbance maximum of 661 nm, range: 590-720 nm), BBQ-650® (absorbance maximum of 650nm, range:550-750nm), Iowa Black® FQ (absorbance maximum of 531nm, range:420-620nm), and Iowa Black® RQ (absorbance maximum of 656nm, range: 500-700nm).

[0108] In another embodiment, the barcode sequence may directly hybridize to the target sequence without the primary complement strand (e.g., ISH probe) having hanging barcode sequence. In such a case, the quencher strand-imager strand hybridization region is kept at one end of the barcode (with or without an addition of a short length of a spacer sequence between the barcode sequence and hybridization sequence) and the toehold region is hanging on the other end of the barcode, thus keeping all the features of the invention.

[0109] Further, although the probe in Figure 2 is utilized here just for the test of DNA sequence kinetics, which is composed of a universal fluorescent imager (domain d*), a holder strand (domains b, c, and d), a bridge strand (domains c* and a*) that hybridizes with the barcode (domain a) appended to an ISH probe for a target RNA (or DNA), it might be utilized for other purposes to economize the costs for the conjugation of fluorophore. For example, all targets can be labeled Attorney Docket no. 10457-596PC0 with fluorescence of such probe complexes at the beginning, and the fluorescence of each target is removed one by one by sequential addition of displacer strands (domains c* and b*), and the reduced fluorescence intensity is calculated after each displacer addition, which may be converted to the fluorescence intensity allocated to a specific target.

[0110] EXAMPLES

[0111] Example 1, Materials and Methods

[0112] 1.1. Cell culture

[0113] U2OS cells were grown in DMEM medium (Gibco, catalog no. 10569) supplemented with 10% (v / v) fetal bovine serum (Gibco, catalog no. 10082), 100 U m-1penicillin, and 100 pg ml-1streptomycin. The cells were cultured at 37 °C in the presence of 5% CO2. For washing and passaging, 1 * DPBS (Gibco, catalog no. 14190) and 0.05% trypsin (Gibco, catalog no. 25300) were used.

[0114] 1.2. Design of a target-hybridization fragment having a barcode sequence

[0115] All target-hybridization fragments (i.e., in situ hybridization probe or ISH probe) having barcode sequences were ordered in desalted form from Integrated DNA Technologies (IDT).

[0116] All target-hybridization fragments having barcode sequences (i.e., ISH probe) for each RNA transcript were combined in an equimolar mixture at 2 pM total in RNAse- and DNAse-free ultrapure water (Invitrogen, catalog no. 10977) and stored at -20 °C until use.

[0117] 1.3. Design of probe having toehold regions (toehold probe)

[0118] The fluorophore-labeled imager strands and the quencher-labeled quencher strands were ordered with HPLC purification from IDT. BHQ-3, BHQ-2, or Iowa black RQ was used for Atto-647, Atto-565, or Atto-488 quenching. Iowa black FQ was used for Alexa-647 quenching. The quencher and imager strands were mixed in l x PBS buffer (Invitrogen, catalog no. 00818654) at a ratio of 1.2:1 (quencher strand to imager strand) to prepare probes with a final probe concentration of 250 nM. The quencher strand is in excess to ensure complete quenching of imager strands. Attorney Docket no. 10457-596PC0

[0119] 1 .4. Sample fixation and permeabilization

[0120] U2OS cells with a concentration of 2 x 105cells per milliliter were cultured on a smart substrate (Interherence, catalog no. SmS-R-16) in an incubator (37 °C with 5% CO2) overnight. Culture medium was aspirated, and cells were rinsed in 1 * PBS buffer. Cells were then fixed in a solution containing l x PBS buffer with 4% paraformaldehyde (v / v) (Electron Microscopy Sciences, catalog no. 191203) for 10 min, and fixation was then quenched with a solution of 100 mM NH4CI for 5 min.

[0121] After fixation, samples were rinsed once in lx PBS, permeabilized in l x PBS with 0.5% Triton X-100 (Sigma, catalog no. SLBV4122) for 10 min and washed once in 2x SSCT buffer (2x SSC buffer (Ambion, catalog no. 1501001) with 0.1% Tween-20 (Sigma, catalog no. SLCB0668)).

[0122] 1.5. RNA FISH and DAPI staining

[0123] Target-hybridization fragments having barcode sequences were added to the sample in in situ hybridization solution comprising 2x SSCT, 50% formamide (v / v) (Sigma, catalog no. S4117), 10% dextran sulfate (Sigma, catalog no. 42867) and the target-hybridization fragments at 100 nM in total concentration. Samples were placed on a flat-block thermocycler, denatured at 60 °C for 3 min, and then incubated at 46.5 °C overnight.

[0124] After incubation, 250 pl 2x SSCT with 10% formamide (VWR, catalog no. EM-4610), which was prewarmed to 60 °C, was added to the sample, followed by aspiration. Samples were then washed with 2x SSCT with 10% formamide at 60 °C for (4 x 5 min). Finally, samples were rinsed once in 2x SSCT.

[0125] For hybridization between the barcode and probe having toehold regions, samples were rinsed once in 1 x PBS buffer, and then preassembled probes having toehold regions of 100 pl at a total concentration of 250 nM (with excess 50 nM of quencher strand to ensure complete quenching of the imager) in l x PBS buffer were added. Samples were incubated at room temperature for 30 min and washed with 1 x PBS buffer at 37 °C for (3 x 3 min).

[0126] Samples were then stained with a solution containing in 1 x PBS buffer with 4,6-diamidino- 2-phenyl indole (DAPI; 0.1 pg ml-1) for 2 min. Finally, samples were transferred to 50 pl of Attorney Docket no. 10457-596PC0 imaging buffer consisting of l x PBS, 1 x protocatechuic acid (Sigma, catalog no. 03930590), l x Trolox and lxprotocatechuic dioxygenase (Sigma, catalog no. 9029-47-4).

[0127] 1.6. Retina tissue preparation and WAG staining

[0128] Retinas were dissected from CD-I mice at postnatal day 18. Retinas were fixed with 4% paraformaldehyde (v / v), 0.25% Triton X (w / v) for 30 min at room temperature and then washed with PBS for 2 x 5 min. The tissue was then cryoprotected by incubation in a 7% sucrose PBS (w / v) solution for 10 min at room temperature, and then transferred to a 1:1 solution of optimal cutting temperature compound (OCT) and 30% sucrose (w / v) in PBS for 1 h. Retinas were then transferred into cryomolds and frozen in the 1:1 solution of OCT and 30% sucrose. Before tissue sectioning, eight-well ibidi chamber or heating substrate were coated with poly-D-lysine (0.3 mg ml-1in 2x borate buffer) for 1 h at 37 °C, and then rinsed twice with water and allowed to dry completely. Embedded retinal tissue was then sectioned on a cryostat in the transverse orientation with a thickness of 14 pm. Sections within ±175 pm of the optic nerve head were collected and mounted on poly-D-lysine coated chamber slides or heating substrate. After washing with lx PBS buffer to remove OCT, 0.5% Triton X-100 in lx PBS buffer was used to penetrate the cells.

[0129] The target-hybridization fragments having barcode sequences in hybridization buffer (2x SSCT, 50% formamide (v / v), 10% dextran sulfate) was incubated with the tissue for overnight hybridization. For single-plex RNA targets, the target-hybridization fragment concentration was 100 nM. For multi-plex RNA imaging, the total target-hybridization fragment concentration was 500 nM. The remaining procedures were the same as the cultured cell processing.

[0130] For the sm-FISH imaging of the RNA targets, only imager strand of the probes at a concentration of 250 nM is applied for imaging. After binding of the probes, wheat germ agglutinin (WGA) conjugated to 405s (Biotium, catalog no. 29027) was diluted to a concentration of 10 pg ml-1in lx PBS to stain the cell membrane of the retina tissue. Retina tissue samples were incubated for 10 min before imaging.

[0131] 1.7. Microscopy imaging

[0132] Samples were imaged on a Nikon Eclipse Ti-E microscope with Plan Apo xlOO oilimmersion objective with numerical aperture of 1.45. The microscope has a Yokogava spinning Attorney Docket no. 10457-596PC0 disk unit (CSU-W1 , Yokogawa Electric) attached, and the excitation lasers (405 nm, 485 nm, 561 nm, 647 nm) are coupled directly into the Yokogawa W 1 unit using a xlOO lens. Images were taken on an EMCCD camera (iXon3, Andor Technologies).

[0133] Retina tissue was imaged with a Yokogawa CSU-W 1 spinning disk confocal unit attached to a fully motorized Nikon Ti2 inverted microscope equipped with a Nikon lincar-cncodcd motorized stage and an Andor Zyla 4.2 plus sCMOS camera using a Nikon Apo xlOO lens. The final digital resolution of the image was 0.16 pm per pixel.

[0134] 1.8. Image processing and analysis

[0135] Maximum Z projections of raw images were processed with ImageJ2. Image analysis including puncta analysis (identification, location and intensity information retrieval) and cell segmentation were conducted with custom MATLAB (R2020a, Mathworks) code. For puncta identification, a signal threshold was applied to the images to generate a binary image. The puncta were then identified, and the location information was retrieved. The puncta intensity was obtained as the maximum value of all the pixels the puncta covered. For cell segmentation, cell masks were created manually and then converted to a binary mask. The binary masks were applied to the original images for single-cell gene expression analysis.

[0136] To count the puncta in retina tissue sample, cells expressing the target RNAs were selected and segmented manually based on the WGA staining in 405 nm channel.

[0137] All individual fluorescent images were generated in Adobe Photoshop or Imagel, and the brightness and contrast were adjusted linearly for display purpose.

[0138] Example 2. Principle of RNA imaging with toehold FISH

[0139] Toehold-FISH leverages programmable, orthogonal, and rapid DNA strand displacement to activate and deactivate fluorescence in situ. In toehold-FISH method, the fluorescent signal of RNA targets is encoded by pre-assembled DNA toehold probes, which comprises a fluorophore- labeled imager strand and a quencher strand that hybridizes to the imager strand to repress fluorescence. Signal activation and deactivation occur through rapid, toehold-mediated displacement reactions using non-fluorescent quencher and imager displacer strands, respectively. Multiplexed RNA imaging is achieved through orthogonal and parallel displacement of reactions. Attorney Docket no. 10457-596PC0

[0140] Because of the vast sequence space of toehold DNA probes, toehold-FISH theoretically enables multiplcxity of thousands of targets, without a limitation in multiplcxity for experimental high- plex imaging .

[0141] At the core of toehold-FISH is the design of toehold probes that enable efficient fluorescence generation and removal (Figure la). Each toehold probe comprises an imager strand with three domains (b*, c*, d*) and a quencher strand with two domains (a, b). The two strands are hybridized via domain b / b*, allowing the quencher to repress the signal of fluorophore through direct quenching39. To target RNAs, ISH probes with a barcode (domain c) bind to specific RNA targets, and the pre-assembled toehold probe hybridizes to the barcode via domain c*. This forms a complex of three strands: the ISH probe, quencher, and imager, with two single-stranded toehold domains (a on the quencher, d* on the imager) available for displacement. Minimal signal is observed because of efficient direct quenching39. All domains are designed to be 18 nucleotides long to ensure stable binding, vast sequence space, favorable thermodynamics, and rapid displacement kinetics.

[0142] In the imaging process, toehold-FISH uses non-fluorescent displacer strands to control fluorescent signal of RNA targets in situ. As shown in Figure la and lb, to activate fluorescence, a quencher displacer strand (domains b* and a*) initiates displacement via the quencher’s toehold domain a, releasing the quencher from the RNA target into the imaging buffer and enabling the imager’s fluorescence. To deactivate fluorescence, an imager displacer strand (domains d and c) binds the imager’s toehold domain d*, displacing the imager off to the solution from the RNA target in situ. As the displacer strands have no fluorescent label, they do not contribute to the background fluorescence when added to the sample. The displaced imager and quencher, present at picomolar concentrations, neither contribute to background fluorescence nor weaken the RNA fluorescent signal. Consequently, the fluidic exchange steps, which are typically required for probe change and background minimization, are eliminated.

[0143] For multiplexed imaging shown in Figure 1c, multiple orthogonal toehold probes bind distinct RNA targets via ISH probes with corresponding barcodes. As displacer strands react only with their corresponding probes, parallel displacement reactions significantly reduce the number of displacement reaction rounds. Except for the first and last rounds, imager displacers (for the previous round’s signal removal) and quencher displacers (for the current round’s signal Attorney Docket no. 10457-596PC0 activation) can be added simultaneously. The order of activation and deactivation is user-defined, and the resulting data can be reconstructed to produce high-plex RNA imaging profiles.

[0144] Example 3. Screening DNA sequences with rapid displacement kinetics for toehold probe designs

[0145] 3.1. Reaction design for kinetic screening

[0146] Although the thermodynamic properties of DNA are well-characterized and modeled40, predicting the kinetics of DNA hybridization, particularly DNA strand displacement41, remains challenging. The kinetic rate of DNA strand displacement varies by orders of magnitude depending on their sequence42, and in situ strand displacement occurs in a fixed and complex cellular environment43, further complicating predictions.

[0147] In this toehold-FISH imaging, each toehold probe undergoes two-step DNA strand displacement reactions to activate and remove the fluorescent signal, respectively.

[0148] To identify DNA toehold probes with rapid kinetics for signal switching, 144 different DNA sequences for displacement reactions were designed and experimentally screened for the final toehold probe designs. (Figures 2 and 3). NUPACK was used to generate 144 different sequences; and ELAVL1 mRNA was used as the target to measure in situ strand displacement kinetics, and the corresponding ISH probes were designed using OligoMiner44’45.

[0149] Since fluorophorc-labclcd DNA strands and in situ ISH probes arc the most expensive reagents, synthesizing 144 fluorophore-labeled strands and ISH probes with different barcodes for experimental evaluation would be very costly. To economically utilize a universal fluorescent imager for screening, as shown in Figure 2b, a universal fluorescent imager (domain d*) and an ISH probe with a barcode (domain a) appended for the Elavall mRNA were designed. A bridge strand (domains c* and a*), a holder strand (domains b, c, and d), and a displacer strand (domains c* and b*) were used to accommodate diverse displacement DNA sequences for strand displacement for economical sequence screening. Each bridge strand consists of two regions: one for binding the imager and another for validating the strand displacement reaction.

[0150] 3.2. Sequence design and fluorescent imaging data collection Attorney Docket no. 10457-596PC0

[0151] All displacement reactions were tested with less than 10% crosstalk. All DNA probes and sequences were ordered from IDT.

[0152] To evaluate displacement kinetics experimentally, a fluorescent DNA probe complex was assembled from the bridge strand, holder strand, and universal imager. Cultured U2OS cells were fixed and permeabilized for primary Elavall ISH probe hybridization, after which the assembled probe complex was applied to the fixed U2OS cells. After washing away excess probe complex, the corresponding displacer strand was added. Fluorescent images of RNA targets were captured using a microscope at 0 s, 10 s, 30 s, 60 s, 90 s, and 120 s after the addition of the displacer DNA strand.

[0153] 3.3. Data processing and kinetic fitting

[0154] RNA puncta were identified in the fluorescent images at 0 s, and their fluorescence intensity and location were recorded. The locations of the RNA puncta at 0 s were used to track their fluorescence signals in subsequent images at 10 s, 30 s, 60 s, 90 s, and 120 s at the same locations. The fluorescence signals of all RNA puncta were averaged at each time point, and first- order kinetic fitting was performed.

[0155] When the displacer DNA was introduced to the sample, the chemical reaction between the displacer DNA and the probe occurred as follows:

[0156] The differential equation for the reaction is: where kdisp is the rate constant, the fluorescence signal observed through a fluorescence microscope is proportional to the [RNA-imager] concentration.

[0157] For a typical mRNA, the abundance is assumed to be 1-10,000 copies per cell, with the sample typically containing 104cells in a 100 pL imaging buffer chamber. Each mRNA is targeted by 48 unique probes. The concentration of bound imager is calculated to be 10'14to 10'12M. As the in-situ RNA-imager concentration (below pM levels) is extremely low compared with the Attorney Docket no. 10457-596PC0 displacer concentration (approximately 1 pM), the displacer concentration is assumed to remain constant throughout the reaction.

[0158] The integral of the differential equation above from Os to time point t gave the following:

[0159] The initial [RNA-Imager]o concentration is proportional to the RNA puncta fluorescence signal at 0 s. The above equation was used to fit the data collected from fluorescence images at different time points, and the rate constant kaspwas derived from the fitted curves. To ensure signal switching was completed within 30 seconds, with 96% of the signal activated or removed, a threshold for the rate constant was set for displacement sequence selection 1.2 x 105s-1. Among 144 unique sequence designs, 62 reactions exhibited a rate constant above this threshold. Example kinetic fittings for DNA sequences with rapid and slow kinetics are shown in Figure 2c, and a scatter plot summarizing the rate constants from all 144 reactions is presented in Figure 2d. Corresponding fluorescence images at different time points are shown in Figure 2e.

[0160] Following the addition of displacer strand, the ELAVL1 mRNA signal decreased as the strand displacement reaction progressed. The time course fluorescent images were taken, and first- order kinetic fitting was performed to determine the rate constant for all the 144 reactions (Figure 3). To ensure rapid kinetics to complete the reaction within 30 seconds, only sequences with a kinetic rate greater than 1.2 x 105s’1were selected for the design of toehold probes (Figure 2). Ultimately, 62 distinct sequences were identified for rapid DNA strand displacement reactions.

[0161] As each toehold probe’s signal activation and removal is achieved through two independent strand displacement reactions, 25 different toehold probes were designed with 50 DNA sequences from screened DNA displacement reaction with rapid kinetics for multiplexed imaging (Table 1). Because of the vast sequence space for the toehold probes, more kinetically rapid toehold probes can be further screened in necessary scenarios.

[0162] Example 4, Validation of toehold FISH with RNA imaging in fixed cells

[0163] Next, the two-step strand displacement reactions for signal activation and removal of the toehold probe were validated by adding quencher and imager displacer DNAs to the sample by imaging RNA in cultured U2OS cells. ELAVL1 mRNA was used as the target (Table 2), pre Attorney Docket no. 10457-596PC0 assembled toehold probes were added to the sample to bind to the ELAVL1 ISH probe via the barcode. The imager is labeled with Atto-647 dye at 5’ end and quencher is labeled with BHQ-3 quencher at 3’ end. After washing off the excess probes, the sample was placed on the microscope stage. Images were captured prior to the addition of any displacers to confirm that the toehold probe's fluorescent signal was fully quenched (Figures 4a and 4b). To activate the toehold probe signal, a standard pipette was used to introduce the quencher displacer into the sample and make a final quencher displacer concentration to be 1 pM. Time-course recordings of signal change following the addition of quencher displacers were conducted to evaluate the speed of signal activation with a spinning disk confocal microscope.

[0164] As shown in Figures 4c, 4g and 4h, the fluorescent RNA puncta showed up clearly after the displacement reaction, and the signal reached its maximum within 30 seconds. Similarly, the kinetic profiling was also performed after the addition of imager displacer after the quencher displacement reaction. The RNA fluorescent signal decreased rapidly within 30 seconds (Figures 4d, 4i, and 4j).

[0165] Standard single molecule FISH (smFISH) experiment was conducted with a sample containing only the imager serving as the control to evaluate the quantities resolved by the toehold- FISH (Figure 4e). As shown in Figure 4k, the single-cell RNA expression of EEAVE 1, measured using smFISH and toehold-FISH, is at the same level, confirming the accuracy of toehold-FISH for quantifying RNA gene expression. Negligible RNA was detected when the toehold probe is inactive or after the imager is displaced.

[0166] Example 5. Rapid and wash-free 25-plex cellular RNA imaging with sequential toehold-FISH using a single fluorophore channel

[0167] In this section, the overall time consumption of the multiplexed imaging workflow was compared using toehold-FISH with fluidic exchange of DNA imagers.

[0168] After sample preparation, the sample is typically placed on the microscope for multiplexed imaging.

[0169] Then, 25-plex RNA imaging was validated using the designed 25 different toehold probes. 25 distinct RNAs, ranging from low to high expression levels, were selected based on bulk RNA sequencing of U2OS cells46. All imagers of toehold probes were labeled with Atto-565 dye, and Attorney Docket no. 10457-596PC0 all quenchers were modified with BHQ-2. Therefore, only the 565nm fluorophore channel was used for the 25-plcx imaging to demonstrate the robustness of the toehold- FISH imaging.

[0170] OligoMiner was used to design the ISH probes, with 25 unique barcodes appended to their 3’ ends for corresponding toehold probes (Table 2). The probe’s binding with barcode was analyzed in silico at 37°C (Figure 5). The 25 different toehold probes were first applied to image their assigned RNA targets individually to confinn their rapid kinetics (Figure 6). The fluorescent signal of all the toehold probe can be activated and removed within 30 seconds (Figure 6). The orthogonality of 25 different toehold probes was further tested, only the corresponding displacer can activate or remove the fluorescent signal (Figure 7).

[0171] To achieve 25-plex RNA simultaneously imaging, all the ISH probe hybridization were applied to the fixed cell for overnight hybridization. A mixture of the pre-assembled 25 toehold probes was then added to bind their respective RNA targets in fixed U2OS cells in a single step. To sequentially activate and removal the toehold probe signal through 25 rounds of displacement reaction, a set of 25 displacer mixtures, each at a concentration of 100 pM, was prepared.

[0172] As shown in Figure 8a, the displacer for first round contained only the quencher displacer for toehold probe 1, the displacer for the final round contained only the imager displacer for toehold probe 25, and the displacer mixtures in the middle round N contained quencher displacer for toehold probe N and imager displacer for toehold probe N-l . The displacer mixtures were sequentially added to the sample to initiate strand displacement, activating the fluorescent signal for the current round and removing the signal from the last round. Each round of strand displacement lasted 30 seconds, followed by fluorescent image capture. The 25-plex RNA images were acquired using a single fluorophore channel at 565 nm in less than 20 minutes (10 seconds for operation, 30 seconds for displacement reaction, 5 seconds for imaging per round) for a single field of view.

[0173] After the registration of all the images across 25 rounds based on nucleus staining with DAPI, the fluorescent images of 25 RNAs in a single representative cell were shown in Figure 8b, clearly depicted all 25 RNA species with each RNA name and assigned toehold probes. The overlap of 25 RNAs in a whole field were shown in Figure 8c, with a white boxed region indicating the cell in Figure 8b. The cells were segmented and all the 25 RNAs were identified to obtain single-cell gene expression profile from the fluorescent images. Attorney Docket no. 10457-596PC0

[0174] The smFISH fluorescent images were collected for each individual RNAs (Figure 9) to obtain their single cell expression level to compare with the 25-plex toehold FISH imaging. As shown in Figure 8d, RNA expression levels obtained from toehold-FISH correlated strongly with those from individually validated smFISH imaging with a slope of 1.02 and correlation of 0.98, confirming that high-plex toehold-FISH reliably quantifies RNA in cultured cell samples.

[0175] The multiplexed imaging workflow consists of three components: (1) signal switch from one target to the next target, (2) movement of the microscope stage across the sample, and (3) laser exposure and image acquisition

[0176] Compared with fluidic-exchange-based multiplexed RNA imaging, toehold-FISH significantly reduced signal switching time from tens of minutes to 30 seconds. To quantitatively compare the time consumption of toehold-FISH and fluidic-exchange workflows, the overall time consumption was calculated as a sum of the three parts under different rounds of imaging and size of imaging area.

[0177] 5.1. Signal switching time

[0178] The signal switching time for toehold-FISH per round, ttoehoid, is 30 seconds, while the signal switching time for fluidic exchange, IFE, is assumed to be 20 minutes25, 26. If n rounds of exchange are required, the total time for signal switching can be calculated as follows: switch— FE- “ (n'FE

[0179] 5.2. The time used for the moving of microscope stage across the sample

[0180] To image a large sample area, tiled imaging is required. Here, it is assumed that a single slice of the sample is imaged along the z-axis. The microscope stage must move between tiles to cover the entire sample area. The sample has a dimension of D, with an imaging area of D2. We assume the microscope is equipped with a camera providing 2304 * 2304 pixels at 6.5 pm per pixel and a 60* objective, typical setup for fluorescent RNA imaging. Thus, the dimension of a single field of view is approximately d = 249.6 pm. The total number of tiles required to cover the sample is calculated as follows: Attorney Docket no. 10457-596PC0

[0181] The average time for the microscope to move is t mi croscope-step 1 second. The total steps that the microscope stage needs to move across all the samples is N-l. Consequently, the total time consumed for the microscope stage to move across the sample area is calculated to be:

[0182] 5.3. Camera imaging time

[0183] When the microscope stage moves to each tile, images are taken by the camera for each tile. The camera imaging time depends on the channel number and exposure time. If three channels are used and exposure time for each channel is assumed to be 500 ms, and the camera imaging time at each tile is tcamera-slep 1.5 s. The total camera imaging time is calculated to be:

[0184] The difference between the toehold-FISH and fluidic exchange workflow is the signal switching time. Therefore, the overall total imaging time for a given sample with a dimension D for the two methods is calculated as the following:

[0185] The time consumption ratio of toehold-FISH to fluidic exchange workflow is calculated as:

[0186] The total time consumption depends on the sample size and the number of exchange rounds. The time consumption ratio for sample sizes ranging from 0.1 to 20 mm and exchange rounds from 1 to 100 were compared. A heatmap of the time consumption ratio is presented in Figure 5a.

[0187] Generally, the larger the number of exchange rounds is, the faster the toehold-FISH workflow is compared with the fluidic exchange workflow. The conditions under which the toehold-FISH workflow is 2-fold, 5-fold, 10-fold, 20-fold, and 30-fold faster than fluidic Attorney Docket no. 10457-596PC0 exchange are shown in Figures 5b, 5c, 5d, 5e, and 5f, respectively, and are indicated as red lines in the hcatmap (Figure 10).

[0188] Example 6. RNA imaging with toehold-FISH in retinal tissues

[0189] Toehold-FISH in complex heterogenous tissue samples were further validated using PFA- fixed mouse retinal tissue. PRKCA mRNA, which serves as a cell type-specific RNA marker for rod bipolar cells located in the inner nuclear layer of retinal tissue (Figure Ila), were used as the target. The ISH probe was designed with a DNA barcode appended at its 3’ end for toehold probe binding. After fixation and permeabilization of the retinal tissue, the ISH probe was incubated overnight to bind PRKCA mRNA, followed by the incubation with the toehold probe that hybridizes to the barcode. The control sample was treated with only the imager binding for smFISH imaging. Before adding the quencher displacer to activate the signal, minimal signal was observed from the retinal tissue sample, indicating high quenching efficiency (Figure 11b). After adding the quencher displacer strand, time-course measurements of fluorescent signal were performed for all RNAs in the field of view.

[0190] As shown in Figure 11c, most of the signal for PRKCA mRNA showed up in the desired inner nuclear layer of the retinal tissue. The averaged time course fluorescent signal for all the RNAs increased and reached a plateau in approximately 60 seconds (Figure lie), indicating reaction completion. Following the addition of the imager displacer, the RNA signal gradually decreased and became undetectable after about 60 seconds, confirming complete signal removal (Figures lid and Ilf). Compared with fixed cultured cells, the displacement reaction in retinal tissue was slower, likely due to the extracellular matrix and high cell density. Retinal tissue is known for its high cell density47, which facilitates external signal reception and transmission to the brain. The rapid signal switching within 1 minute in retinal tissue demonstrated toehold- FISH’s broad applicability to tissue samples. The resolved single cell RNA copy number of PRKCA with toehold-FISH is at the same level as the one resolved by smFISH, indicating reliability of getting quantitative expression information in tissue samples (Figure 11g).

[0191] Example 7, 24-plex RNA imaging in fresh frozen retinal tissue samples using single fluorophore channel Attorney Docket no. 10457-596PC0

[0192] The robustness of toehold-FISH in tissue samples supports its application for high-plex RNA imaging to resolve single cell types in complex tissue samples. The retina consists of -130 different cellular subtypes which are categorized into 7 different cell classes. This immense cellular diversity is arranged histologically into three distinct layers and work together to extract and process different visual features (Figure 12a). For instance, bipolar cells are a class of interneuron located in the inner nuclear layer and pass information from the photoreceptors in the outer nuclear layer to the retinal ganglion cells in the ganglion cell layer that transmit this processed information to the brain48. In mice, there are 15 different subtypes of bipolar cells which process different visual features and can be identified molecularly49.

[0193] To demonstrate the toehold FISH performance for cell typing in heterogenous and complex tissue structure, 24 different RNA markers for various cell types in the retina are selected for 24- plex toehold-FISH imaging. OligoMiner was used to design ISH probe for all the RNAs and the barcodes for 24 different toehold probes are appended (see Table 2). After the primary ISH probe and toehold probe binding, the retinal tissue sample was placed on the microscope stage. WGA labeled CF405s dye were used to stain the cell membrane. The prepared 24 displacer mixtures were sequentially added to the sample for toehold probe signal activation and removal, and imaging was taken after 1 minute of reaction for each round (Figure 12b). The whole workflow was completed within 30 minutes (10 seconds for operation, 60 seconds for reaction, 5 seconds for imaging per round) for the 24 rounds imaging to visualize 24 different RNAs for a single field of view.

[0194] The individual 24 RNA images were shown in Figure 12c (see Figure 13 for individual fluorescent images of 24 RNAs). All the RNAs were clearly visualized in their desired region in the retinal tissue. Based on the 24-plex RNA expression, it was possible to clearly resolve different cell types (Figure 12d), such as rod photoreceptor cells with Cngal, amacrine cells with Glytl, and ganglion cells with Nefm50, 51. Several sub-type of bipolar cells in the intemuclear region was also identified, such as type 2 bipolar cells with Neto2, type 5c with Slitk5, type 5d with Meis2, type 7 with Igfnl49, 52. The resolved single cell RNA expression data (Figure 12c) were further compared with smFISH (Figure 14), confirmed consistency between the two methods for both spatial location and resolved single cell expression.

[0195] Example s. DNA Sequences Attorney Docket no. 10457-596PC0

[0196] Table 1 . DNA Sequences of the Barcodes

[0197]

[0198] Attorney Docket no. 10457-596PC0

[0199] References.

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Claims

1. Attorney Docket no. 10457-596PC0What is claimed is:

1. A DNA toehold probe (TP) for fluorescence in situ hybridization (FISH), which comprises an imager strand and a quencher strand, wherein the imager strand is conjugated with a fluorophore, and the quencher strand with a quencher; wherein the imager strand binds to a barcode sequence; wherein the fluorescence of the imager strand is detected when the quencher strand is removed from the imager strand by strand displacement initiated at the toehold region of the quencher strand by a quencher displacer strand, and wherein the fluorescence of the imager strand is removed from the target when the imager strand is removed by strand displacement initiated at the toehold region of the imager strand by an imager displacer strand.

2. The DNA toehold probe of claim 1, wherein the barcode is a unique nucleotide sequence for each target RNA and appended to the 3’ end (or 5 ’-end) of an in situ hybridization probe (ISH probe) specific for a target RNA or DNA.

3. The DNA toehold probe of claim 1, wherein the imager strand has three domains, from the 5’ to 3’ (or 3’ to 5’), b*, c*, and d*, wherein the b* domain is conjugated with a fluorophore at its 5’ end (or 3’ end), and hybridizes to the quencher strand, and; wherein the c* domain hybridizes to the barcode of an ISH probe; and wherein the d* domain is a single-stranded toehold region for an imager displacer strand.

4. The DNA toehold probe of claim 1, wherein the quencher strand has two domains, from the 5’ to 3’ (or 3’ to 5’), a and b, wherein the a domain is a single- stranded toehold region for a quencher displacer strand; andAttorney Docket no. 10457-596PC0 wherein the b domain is conjugated with a quencher at its 3’ end (or 5’ end), and hybridizes to the b* domain of the imager strand.

5. The DNA toehold probe of claim 1 or 4, wherein the quencher displacer strand has two domains (b*, a*), which are complementary to the two domains (b, a) of the quencher strand.

6. The DNA toehold probe of claim 1 or 3, wherein the imager displacer strand has two domains (d, c), which are complementary to the two domains (d*, c*) of the imager strand.

7. The DNA toehold probe of any of the preceding claims, wherein all domains have a nucleotide length in a range of 16-24 nucleotides, optionally about 18 nucleotides.

8. The DNA toehold probe of claim 1 or 2, wherein the fluorophore conjugated to the 5’ end (or 3’ end) of the imager strand is one selected from Atto-647, Atto-565, Atto-488, or Alexa-647.

9. The DNA toehold probe of claim 1 or 3, wherein the quencher conjugated to the 3’ end (or 5’ end) of the quencher strand, which quenches the selected fluorophore of claim 7, is BHQ-3, BHQ-2, Iowa black RQ, or Iowa black FQ.

10. The DNA toehold probe of claim 1 , wherein the probe (TP1) comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:51 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:26, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 101 and SEQ ID NO:76, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 1.

11. The DNA toehold probe of claim 1, wherein the probe (TP2) comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:52 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:27, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 102 and SEQ ID NO:77, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 2.Attorney Docket no. 10457-596PC012. The DNA toehold probe of claim 1 , wherein the probe (TP3) comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:53 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:28, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 103 and SEQ ID NO:78, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 3.

13. The DNA toehold probe of claim 1, wherein the probe (TP4) comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:54 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:29, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 104 and SEQ ID NO:79, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 4.

14. The DNA toehold probe of claim 1, wherein the probe (TP5) comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:55 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:30, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 105 and SEQ ID NO:80, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 5.

15. The DNA toehold probe of claim 1, wherein the probe (TP6) comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:56 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:31, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO:106 and SEQ ID NO:81, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 6.

16. The DNA toehold probe of claim 1, wherein the probe (TP7) comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:57 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:32, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 107 and SEQ ID NO:82, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 7.Attorney Docket no. 10457-596PC017. The DNA toehold probe of claim 1 , wherein the probe (TP8) comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:58 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:33, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 108 and SEQ ID NO:83, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 8.

18. The DNA toehold probe of claim 1, wherein the probe (TP9) comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:59 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:34, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 109 and SEQ ID NO:84, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 9.

19. The DNA toehold probe of claim 1, wherein the probe (TP 10) comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:60 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:35, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 110 and SEQ ID NO:85, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 10.

20. The DNA toehold probe of claim 1, wherein the probe (TP11) comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:61 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:36, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 111 and SEQ ID NO: 86, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 11.

21. The DNA toehold probe of claim 1, wherein the probe (TP 12) comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:62 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:37, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 112 and SEQ ID NO: 87, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 12.Attorney Docket no. 10457-596PC0 l. The DNA toehold probe of claim 1 , wherein the probe (TP 13) comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:63 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:38, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 113 and SEQ ID NO:88, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 13.

23. The DNA toehold probe of claim 1, wherein the probe (TP 14) comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:64 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:39, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 114 and SEQ ID NO:89, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 14.

24. The DNA toehold probe of claim 1, wherein the probe (TP15) comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:65 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:40, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 115 and SEQ ID NO:90, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 15.

25. The DNA toehold probe of claim 1, wherein the probe (TP 16) comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:66 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:41, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 116 and SEQ ID NO:91, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 16.

26. The DNA toehold probe of claim 1, wherein the probe (TP17) comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:67 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:42, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 117 and SEQ ID NO: 92, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 17.Attorney Docket no. 10457-596PC027. The DNA toehold probe of claim 1 , wherein the probe (TP18) comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:68 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:43, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 118 and SEQ ID NO:93, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 18.

28. The DNA toehold probe of claim 1, wherein the probe (TP 19) comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:69 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:44, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 119 and SEQ ID NO:94, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 19.

29. The DNA toehold probe of claim 1, wherein the probe (TP20) comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:70 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:45, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 120 and SEQ ID NO:95, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 20.

30. The DNA toehold probe of claim 1, wherein the probe (TP21) comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:71 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:46, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 121 and SEQ ID NO:96, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 21.

31. The DNA toehold probe of claim 1, wherein the probe (TP22) comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:72 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:47, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 122 and SEQ ID NO:97, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 22.Attorney Docket no. 10457-596PC032. The DNA toehold probe of claim 1 , wherein the probe (TP23) comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:73 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:48, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 123 and SEQ ID NO:98, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 23.

33. The DNA toehold probe of claim 1, wherein the probe (TP24) comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:74 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:49, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 124 and SEQ ID NO:99, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 24.

34. The DNA toehold probe of claim 1, wherein the probe (TP25) comprises an imager strand comprising a nucleotide sequence of SEQ ID NO:75 and a quencher strand comprising a nucleotide sequence of SEQ ID NO:50, wherein the matching imager displacer and quencher displacer for strand displacements comprise nucleotide sequences of SEQ ID NO: 125 and SEQ ID NO: 100, respectively, and wherein the barcode sequence matching the c* domain of the imager strand is SEQ ID NO: 25.

35. A kit comprising any one of the toehold probes according to claims 10-34 and its matching quencher displacer / imager displacer pair, wherein the kit comprises imager strands labeled with a fluorophore, quencher strands, imager displacer strands, quencher displacer strands, and a small brochure introducing a matching barcode DNA sequence.

36. A package comprising multiplex of the kit of claim 35, wherein the fluorophore of each imager strand is the same or a different fluorophore, and wherein the package may further comprise a concentrated imaging buffer solution to be 5-50 times diluted for use.

37. Multiplexed toehold-FISH imaging method, comprising steps of i. fixing and permeabilizing a sample, which can be cells cultured on a coverslip or thin-sliced paraffin-embedded tissues,Attorney Docket no. 10457-596PC0 ii. labeling multiplex RNA or DNA targets with multiplex ISH probes, wherein each ISH probe has non-hybridizing unique barcode sequence at one end; iii. assembling toehold probes; iv. labeling multiplex barcodes with multiplex toehold probes; v. performing DAPI nuclei staining; vi. washing the sample, adding imaging buffer to the sample, and setting up the sample on the microscope stage; vii. measuring the basal level of fluorescence; viii. adding a quencher displacer strand for a toehold probe; ix. after incubating the sample for a few minutes (1-5 minutes) at 20-30°C, measuring fluorescence; x. adding an imager displacer strand for the toehold probe; xi. repeating the steps of vii-x and determining the level of fluorescence for each target by subtracting the basal level of fluorescence measured at step vii from the fluorescence measured at step ix; wherein optionally in the middle round n between the first round and the last round, the quencher displacer of the nth target (step viii of the nth round), and the imager displacer of the (n-7)th target (step x of the (?7-7)th round) can be added together as a mixture (for parallel displacement reactions), and in step xi, the fluorescence signal for each target is determined by subtracting the basal level of fluorescence measured at the first round of step vii from the fluorescence measured at each round step ix.

38. The multiplexed toehold-FISH imaging method of claim 37, wherein the toehold probe (TP) is at least one selected from TP1-TP25 according to claims 10-34.

Images